Neurotrophic factors control differentiation and survival of neural cells and, thereby, play an essential role in the development and maintenance of the nervous system. In Project 2, we will test the hypothesis that neurospheres include multipotential cells that can be directed down multiple lineages by neurotrophic factors. Regulation of multipotential cells by neurotrophic factors may be one mechanism by which the nervous system produces the optimum number of each t ype of neural cell. In preliminary studies, we have demonstrated that neurosphere cells express receptors for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3) and ciliary neurotrophic factor (CNTF). Each of these factors enhances differentiation of neurosphere cells. NGF and BDNF enhance neuronal differentiation, and NT-3 and CNTF enhance both neuronal and oligodendroglial differentiation. Astroglial differentiation is induced by epidermal growth factor (EGF) deprivation. Based on these results, we propose the following Specific Aims: (1) Determine whether the differentiated cells derived from neurosphere cells have the attributes of functional neurons. We will characterize expression of neuronal markers, neurotransmitter phenotypes, and electrophysiological properties. (2) Analyze the mechanisms by which neurosphere cells differentiate in response to neurotrophic factors. We will determine whether neurotrophic factors induce neurosphere cells to differentiate or enhance survival of cells differentiating in response to EGF deprivation. We will analyze neural precursor cells isolated by Project 1 for neurotrophic factor receptor expression and responsiveness to neurotrophic factors. As a Pilot Experiment, we will test the responsiveness of neurosphere cells isolated from different days of embryonic development and from postnatal and adult mice. (3) Test additional signals and cues that might induce or regulate differentiation of neurosphere cells. We will determine whether neurosphere cells synthesize neurotrophic factors. We will test the influence of local developmental cues by coculturing neurosphere cells with brain cultures and, in collaboration with a project of transplant differentiated cells into murine brain. The effects of nitric oxide donors alone or in conjunction with neurotrophic factors will be determined. These studies are possible only because of our team effort, allowing close interactions and collaborations with other members of the Neuro-Oncology Group.